In correlative light and electron microscopy, the light-microscopic imaging is used to seek in a targeted manner sample points which are subsequently intended to be imaged in the electron microscope with a very high magnification and resolution. In the case of two-dimensional images, this correlation can be achieved by the transmission of 2D coordinates from the light microscope to the electron microscope.
However, it is often desired to examine and image the three-dimensional structure of a sample. This is possible with the aid of tomographic methods, in which individual planes of a three-dimensional sample are imaged without superposition. The three-dimensional structure of the sample can then be represented in a 3D reconstruction with the aid of a series of such slice images.
However, the application of tomography in correlative microscopy is connected with difficulties. Although a confocal light microscope can create a 3D image of a three-dimensional sample if the sample is optically transparent, the slices, imaged thus, situated in the interior of the sample are, however, not accessible to imaging via an electron microscope since the sample is generally not transparent to electrons. Therefore, a coordinate transfer of the 3D data obtained in the light microscope to the electron microscope is connected with difficulties and not very expedient.
Moreover, the sample is usually be embedded before observation in the electron microscope in order to make it stable in a vacuum. However, as a result, the sample changes so strongly in terms of the spatial extent thereof that sample points which are defined by three-dimensional coordinates cannot be retrieved without problems. Furthermore, further preparation steps are usually involved for preparing the imaging by electron microscopy, such as e.g. the contrasting of biological samples with heavy metals.
Although it is feasible to fix and embed the sample prior to the examination by light microscopy so that stable coordinates which no longer change between observation by light microscopy and observation by electron microscopy can be defined, this results in heavy metal dyeing already having to be undertaken during the embedding step. This in turn is disadvantageous in that the state of the sample is not ideal for the examination by light microscopy since the light-optical transparency is reduced and the sample material is no longer suitable for fluorescence examinations.
Another solution approach consists in dissecting the sample into sections of the series. The sample is embedded in advance and dissected into electron-transparent ultrathin sections. Here, the sample sections are so thin that the structures thereof have a virtually two-dimensional form. The acquired 2D coordinates are then transmitted from the light microscope to the electron microscope. There is no transmission of 3D data since the three-dimensionality of the data only emerges from combining a plurality of 2D data records to form a 3D reconstruction. Further disadvantages include that highly precise ultramicrotomes with high quality diamond blades are involved and the axial resolution is limited by the section thickness that can be achieved with the employed ultramicrotome. Conventionally, a section thickness of less than 50 nm to 30 nm cannot be achieved by ultramicrotomes.
Methods in which a sample is dissected into sections of the series are known. The sample sections are deposited on a sample carrier in such a way that the sequence thereof is maintained in the section series. For this purpose, the sample sections are arranged in connected chains or so-called “arrays”. It is possible to create a 3D reconstruction of the sample on the basis of the images of the ordered sample sections. A disadvantage of this is that the sample is cut into ultrathin slices, i.e. into sections, the thickness of which is at most approximately 200 nm. Moreover, very many or almost all sample sections are imaged and screened when searching for regions of interest. This means that a number of sample sections which contain no structures of interest are also imaged and examined with much expenditure of time.
Processes from correlative microscopy, in which the position of a region of interest (ROI) is defined in one microscope and re-approachable in a further microscope by the transmission of 2D data, are also known. A disadvantage here is that this only succeeds for two-dimensional data, ultrathin sections are used and the resolution in the z-direction is limited by the thickness of the sample sections. By contrast, a resolution in the z-direction of a few nanometers is desirable.
The following documents are known: US 2008/0152207A1; WO 2012/080363 A1; Maco B et al. (2013): “Correlative in Vivo 2 Photon and Focused Ion Beam Scanning Electron Microscopy of Cortical Neurons”, PLOS ONE, Vol. 8: Issue 2; and Robinson, J. M. et al. (2001): Correlative Fluorescence and Electron Microscopy on Ultrathin Cryosections: Bridging the resolution Gap, The Journal of Histochemistry and Cytochemistry, 49 (7): 803-803.